The lysosome is an organelle found in the cytoplasm of animal cells that contains more than 50 hydrolases that break down biomolecules during the recycling of worn-out cellular components or after the engulfment of viruses and bacteria. This organelle contains several types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases and sulfatases. All enzymes are acid hydrolases.
Lysosomal storage diseases (LSDs) are caused by genetic defects that affect one or more lysosomal enzymes. These genetic diseases result generally from a deficiency in a particular enzyme activity present in the lysosome. To a lesser extent, these diseases may be due to deficiencies in proteins involved in lysosomal biogenesis.
LSDs are individually rare, although as a group these disorders are relatively common in the general population. The combined prevalence of LSDs is approximately 1 per 5,000 live births. See Meikle P, et al., JAMA 1999; 281:249-254. However, some groups within the general population are particularly afflicted by a high occurrence of LSDs. For instance, the prevalence of Gaucher and Tay-Sachs diseases in descendants from Jewish Central and Eastern European (Ashkenazi) individuals is 1 per 600 and 1 per 3,900 births, respectively.
Type II mucopolysaccharidoses (MPSII), known also as Hunter syndrome and first described by Dr. Charles Hunter, is a chronic, progressive and multisystemic LSDs caused by deficiency or absence of activity of the iduronate-2-sulfatase (IDS) enzyme, encoded by the IDS gene and involved in the lysosomal stepwise degradation of the glycosaminoglycans (GAG) heparan sulfate (HS) and dermatan sulfate (DS), leading to their pathological accumulation. See Hunter, Proc R Soc Med. 1917; 10 (Sect Study Dis Child): 104-16. Due to the X-linked recessive inheritance, almost all Hunter patients are males, although some women with Hunter syndrome have been reported in the literature. See Mossman et al., Arch Dis Child. 1983; 58.911-915, Gullén-Navarro et al., Orphanet J Rare Dis. 2013; 25(8):92, Valstar et al., J. Inherit. Metab. Dis. 2008; 31(2):240-52.
MPSII is characterized clinically as a childhood-onset, progressive neuropathy of the Central Nervous System (CNS). Hunter children are usually normal at birth and develop symptoms before the age of 2 years. See Schwartz et al., Acta Paediatr Suppl. 2007; 96:63-70. The clinical course generally begins with slow-progressive cognitive impairment followed by behavioural problems and progressive intellectual decline. Loss of locomotion occurs later. In addition to the neurological symptoms, MPSII patients suffer from non-neurological alterations, including recurrent ear, nose, throat and chest infections, frequent diarrhoea and constipation, cardiac failure, coarse facial features, short stature, progressive joint stiffness and degeneration, skeletal abnormalities which affect mobility, as well as hepato and splenomegaly. See Neufeld and Muenzer, “The Mucopolysaccharidoses” in Scriver C, et al., Eds., “The metabolic and molecular basis of inherited disease”, McGraw-Hill Publishing Co., New York, N.Y., US, 2001, pp. 3421-3452. The spectrum of clinical manifestations of the disease varies considerably depending on the residual levels of IDS activity that the patient has, which in turn is determined by the underlying mutation of the IDS gene, with >300 mutations of the IDS gene described to date. In general, two clinical forms of MPSII have been described. The most severe form, with an onset between 18 months and 4 years of age, is three times more common than the mild form, and, is characterized by coarse facial features, skeletal deformities, hepatosplenomegaly and neurological involvement which leads to mental retardation. See Wraith et al., Eur J Pediatr. 2008; 167(3):267-277. Patients usually die during the second decade of life due to obstructive airway disease and cardiac failure. See Wraith et al., Eur J Pediatr. 2008; 167(3):267-277, Neufeld and Muenzer, supra. A more slowly progressive form of the disease, with later onset, longer survival and minimal neurological dysfunction, known as the attenuated phenotype, has also been reported in a subset of MPSII patients. See Wraith et al., Eur J Pediatr. 2008; 167(3):267-277, Neufeld and Muenzer, supra.
Until recently there were no specific approved therapies for MPSII syndrome and the only treatment available was symptomatic using a wide range of unspecific drugs for the prevention and management of disease complications. In the last few years, two main therapeutic options have become available: Enzyme Replacement Therapy (ERT) and hematopoietic stem cell transplantation (HSCT). The design of both therapeutic strategies relies on the possibility of cross-correction, based on the fact that normal cells secrete significant amounts of mannose-6-phosphate (M6P)-tagged soluble lysosomal enzymes, such as IDS, which can be subsequently taken up from the extracellular compartment by other cells via M6P receptors on the plasma membrane and targeted to the lysosomes. See Enns et al., Neurosurg Focus. 2008; 24(3-4):E12. In addition, there is a threshold of residual enzymatic activity, generally very low, above which the cell is capable of coping with substrate influx and subjects are not affected by the disease, suggesting that restoration of normal activity is not a requisite to modify the clinical course. See Neufeld, Annu Rev Biochem. 1991; 60:257-80.
Since its approval by the Food and Drug Administration (FDA) in 2006 and by the European Medicines Agency (EMA) in 2007, recombinant human iduronate-2-sulfatasa (Idursulfase, ELAPRASE®, Shire Pharmaceuticals) has been indicated for the treatment of patients with MPSII. The treatment is administered weekly at a dose of 0.5 mg/kg by intravenous infusion, with an average infusion time of 1-3 hours. See Giugliani et al., Genet Mol Biol. 2010; 33(4):589-604. ELAPRASE® was approved after a randomized, double-blind, placebo-controlled study of 96 Hunter patients with no cognitive decline at baseline and with moderately advanced disease. See Muenzer et al., Genet Med. 2006; 8(8):465-73, Muenzer et al., Genet Med. 2011; 13(2):95-101. After one year of treatment, ELAPRASE®-treated patients showed an increase in the distance walked in six minutes (six-minute walk test) compared to patients on placebo. See Muenzer et al., Genet Med. 2011; 13(2):95-101. ERT with ELAPRASE® has also been shown to improve joint range of motion (ROM) and to reduce liver and spleen volumes. See Muenzer et al., Genet Med. 2011; 13(2):95-101. Furthermore, there is evidence of improved pulmonary function when neutralizing antibodies against Idursulfase are not present; development of anti-IDS antibodies was reported in 50% of the long-term treated patients. See Muenzer et al., Genet Med. 2011; 13(2):95-101.
A phase I/II study in 31 MPSII patients compared the efficacy of ELAPRASE® with that of a second product based on the beta isoform of Idursulfase with a proposed commercial name of Hunterase® (NCT01301898). Both proteins were administered intravenously at a dose of 0.5 mg/kg/week for ELAPRASE® and 0.5 and 1.0 mg/kg/week for Hunterase® during 24 weeks. The results from Hunterase® treatment showed reduced urine GAG excretion and improved performance in the 6-minute walking test, but none of the doses was able to mediate therapeutic efficacy in pulmonary function, cardiac function or joint mobility. See Sohn et al., Orphanet J Rare Dis. 2013; 8:42. Hunterase® infusions were generally safe and well-tolerated, although a few adverse events, such as urticaria and skin rash, were reported. See Sohn et al., Orphanet J Rare Dis. 2013; 8:42. A pivotal Pill study has recently been completed (NCT01645189), but results are not yet available.
Due to hypersensitivity to ELAPRASE®, medical support has to be available during product administration. During the trial, the most severe adverse events described were anaphylactic reactions that could appear anytime during ELAPRASE® infusion or up to 24 hours after product administration. See Muenzer et al., Genet Med. 2006; 8(8):465-73, Muenzer et al., Genet Med. 2011; 13(2):95-101. These anaphylactic reactions, that can compromise the patient's life, include respiratory distress, hypoxia, hypotension, urticaria and/or angioedema of throat or tongue and may require interventions such as resuscitation or emergency tracheotomy, and treatment with inhaled beta-adrenergic agonists, epinephrine or intravenous corticosteroids. See Burton et al., Mol Genet Metab, 2011; 103(2):113-20. Other disadvantages of ERT include: 1) the difficulty of performing 1-3 hour-long intravenous infusions in paediatric patients, many of whom suffer from mental illness the fact that 50% of patients treated with ELAPRASE® in clinical studies became positive for antibodies to Idursulfase of yet unknown clinical significance, but which might limit product efficacy in the long-term, as suggested by tests of pulmonary function. See Muenzer et al., Mol Genet Metab. 2007; 90(3):329-37, Muenzer et al., Genet Med. 2006; 8(8):465-73, Muenzer et al., Genet Med. 2011; 13(2):95-101, and 3) the high cost of the therapy, which includes also the costs of home-care. See Wyatt et al., Health Technol Asses. 2012, 16(39):1-543.
Regardless of the safety concerns or the cost of ELAPRASE® administration, the inability of intravenously administered recombinant IDS to reach the CNS, at least at the currently recommended dose of 0.5 mg/kg per week, likely limits the potential applicability of ERT to the treatment of the severe neurodegeneration observed in Hunter patients. Only a partial rescue of IDS brain activity was achieved by weekly intravenous administration of 1.2 or 10 mg ELAPRASE®/kg to 2 or 7 month-old MPSII mice, respectively. See Polito et al., Hum Mol Genet. 2010; 19(24):4871-85. Furthermore, even at these high doses, IDS activity in circulation returned to pre-treatment levels 72 hours post-administration of the protein. See Polito et al., Hum Mol Genet. 2010; 19(24):4871-85. Indeed, intravenous ERT failed to correct GAG accumulation in the brains of a murine model of MPSII. See Garcia et al., Mol Genet Metab. 2007; 91(2):183-90. Therefore, the indication of ELAPRASE® is limited to the treatment of non-neurological symptoms of the disease.
An alternative to the intravenous delivery of ERT is the provision of the exogenous enzyme directly to the CNS. The administration of 20 μg of recombinant human IDS to the lateral ventricle of 5-month-old MPSII mice every 3 weeks increased IDS activity in cerebrum, cerebellum and somatic organs, such as liver, heart, kidney and testis. See Higuchi et al., Mol Genet Metab. 2012; 107(1-2):122-8. The restoration of IDS activity led to the recovery of short-term memory and locomotor activity and to a reduction in cellular vacuolation and lysosomal distension in cerebellum, liver and testis. However, therapeutic efficacy was partial, GAG content was not completely normalized and some behavioural alterations remained refractory to the treatment. See Higuchi et al., Mol Genet Metab. 2012; 107(1-2):122-8. A recent safety and dose ranging study of administration of Idursulfase to the cerebrospinal fluid (CSF) via an intrathecal drug delivery device to directly treat CNS pathology in Hunter patients has demonstrated reductions of approximately 80-90% in CSF GAG levels after 6 months of treatment. See Muenzer et al., Genet. Med. 2015; doi:10.1038/gim.2015.36 and www.clincialtrials.gov (NCT00920647). However, the implantation of the permanent intrathecal delivery device that the therapy requires is associated with substantial risks and shortcomings and the therapy itself has a very high economic cost per patient/year.
Another way to reach the CNS by systemic administration is using a molecular Trojan horse. An example of that approach it the insulin Receptor Antibody-Iduronate 2-Sulfatase fusion protein (HIRMAb-IDS), which can cross the blood-brain barrier (BBB) via receptor-mediated transport. Intravenous administration of 3, 10 and 30 mg/kg of HIRMAb-IDS to male juvenile Rhesus monkeys weekly, for 26 weeks, resulted in a HIRMAb-IDS brain uptake of 1% of the total injected dose. See Boado et al., Biotechnol Bioeng. 2014; 111(11):2317-25. The study also demonstrated safety of the fusion protein, as no infusion-related reaction or immune response was observed.
Hematopoietic stem cell transplantation (HSCT) using bone marrow-derived stem cells (Bone marrow transplantation, BMT) has proven efficient in the treatment of both somatic and neurological pathology in patients with other MPSs. See Peters et al., Blood. 1996; 87(11):4894-902, Peters and Steward, Bone Marrow Transplant. 2003; 31(4):229-39 and Yamada et al., Bone Marrow Transplant. 1998; 21(6):629-34. The principle underlying the correction by HSCT is that donor monocytes are able to cross the capillary wall, even at the blood-brain barrier, after which they differentiate into tissue macrophages, microglia in the case of the CNS, and secrete the deficient enzyme for delivery to the various cells. See Krivit et al., Cell Transplant. 1995; 4(4):385-92. BMT performed in MPSII mice reduced GAG accumulation in a variety of somatic tissues, including liver, spleen and lung, but not in the CNS. See Akiyama et al., Mol Genet Metab. 2014; 111(2):139-46. When BMT is combined with ERT (0.5 mg Idursulfase/kg/weekly), an additive effect on GAG levels in heart, kidney and lung was observed 7 months after treatment of MPSII mice, but accumulation of GAGs in the CNS remained at pathological levels. See Akiyama et al., Mol Genet Metab. 2014; 111(2):139-46. However, the evidence for clinical efficacy is not very strong in MPSII patients. The follow-up of 10 Hunter patients who received BMT between 1982 and 1991 showed highly varying degrees of success. See Vellodi et al., J Inherit Metab Dis. 1999; 22(5):638-48. Four of those patients died before 100 days post-BMT, and 3 more before 7 years after the procedure. Of the 3 patients that survived for more than 7 years after BMT, one of them reported no clinical benefit; a second showed a minimal increase of IDS activity in plasma and the third failed to normalize GAG content despite having a slight increase in IDS activity in plasma. See Vellodi et al., J Inherit Metab Dis. 1999; 22(5):638-48. The Magnetic Resonance Imaging (MRI) of the brain showed a slight decrease in the number of cystic lesions 2.5 years after BMT in a patient with mild MPSII phenotype. See Seto et al., Ann Neurol. 2001; 50(1):79-92. However, the same study provided data on another patient with mild phenotype that did not show any improvement under MRI. See Seto et al., Ann Neurol. 2001; 50(1):79-92. Clinical outcomes appears to be highly variable among Hunter patients, presumably due to various factors; genotype, age at HSCT, patient's clinical status at HSCT, such as degree of neurological impairment, donor status, donor chimerism, stem cell source, and enzyme activity have all been suggested to influence the long-term outcome. See Giugliani et al., Genet Mol Biol. 2010; 33(4):589-604, Valayannopoulos et al., Rheumatology. 2011; 5:v49-59.
When successful, HSCT can contribute to some degree of clinical benefit at somatic level, decrease behavioural problems and better sleeping patterns, but whether the treatment can mediate any significant improvement of cognitive impairment remains unclear. See Giugliani et al., Genet Mol Biol. 2010; 33(4):589-604, Valayannopoulos et al., Rheumatology. 2011; 5:v49-59. In general, this approach is not recommended for Hunter patients, due to the high rate of morbidity and mortality and the variable neurocognitive benefits. See Giugliani et al., Genet Mol Biol. 2010; 33(4):589-604.
A plausible explanation to the failure of HSCT is the limited IDS expression in engrafted cells, leading to an insufficient IDS cross-correction in the CNS. Lentiviral vectors encoding for the human IDS gene were used to transduce bone marrow cells prior to their transplantation into MPSII mice. Treated MPSII mice showed improved performance in the T-maze memory test 14 weeks post-transplant. See Podetz-Pedersen et al., Mol Ther. 2013; 21:s1-s285.
Given the limitations of current therapeutic options for MPSII, alternative approaches are needed. In vivo gene therapy offers the possibility of a one-time treatment for MPSII and other inherited diseases, with the prospect of lifelong beneficial effects. Several gene therapy approaches based on the use of different viral vectors combined with different routes of administration have been tested in animal models of MPSII disease.
Adenoassociated virus (AAV) vector-mediated gene transfer, in particular, is rapidly emerging as the approach of choice for many in vivo gene therapy applications, due to the high transduction efficiency and the lack of pathogenicity of these vectors. AAV vectors can transduce post-mitotic cells and several pre-clinical and clinical studies have demonstrated the potential of AAV vector-mediated gene transfer to efficiently drive sustained expression of therapeutic transgenes for a variety of diseases. See Bainbridge et al., N Engl J Med. 2008; 358(21):2231-9, Hauswirth et al., Hum Gene Ther. 2008; 19(10):979-90, Maguire et al., N Engl J Med. 2008; 358(21):2240-8, Niemeyer et al., Blood 2009; 113(4):797-806, Rivera et al., Blood 2005; 105(4):1424-30, Nathawani et al., N Engl J Med. 2011; 365(25):2357-65 and Buchlis et al., Blood 2012; 119(13):3038-41.
Systemic administration of AAV5-CMV-human IDS vectors to the temporal vein of MPSII mouse pups (p2) resulted in an increase in IDS activity in heart, kidney, liver, lung, muscle and spleen, and a moderated increase in IDS activity in the brain, which led to a reduction in somatic tissue and urinary GAG content up to 18 months post a single vector administration. See Polito et al., Am J Hum Genet. 2009; 85(2):296-301. Also, this treatment prevented the development of CNS pathology by preventing neurodegeneration, and correcting astrogliosis and inflammation. The evaluation of mice in the Open Field Test 18 moths post AAV injection demonstrated the improvement with treatment in the gross motor phenotype of MPSII mice. See Polito et al., Am J Hum Genet. 2009; 85(2):296-301.
AAVs of serotype 8 encoding for the human IDS gene under the control of the liver-specific TBG promoter have also been used to treat MPSII. Up to 7 months following the intravenous administration of vectors to 2 month-old MPSII mice, an increase in serum, liver, spleen, lung, heart, kidney and muscle IDS activity was observed, resulting in complete correction of GAG storage in these somatic tissues. See Cardone et al., Hum Mol Genet. 2006; 15(7):1225-36. However, very high doses (4×1012 viral genomes/mouse) were required to achieve a slight increase in IDS activity and partial clearance of GAG accumulation in the brain when the vectors were administered intravenously. See Cardone et al., Hum Mol Genet. 2006; 15(7):1225-36. Similarly, the intravenous administration of AAV8 vectors in which the human IDS gene is under the control of the ubiquitous elongation factor 1-a (EF) promoter to adult MPSII mice demonstrated an increase in IDS activity in liver, heart, spleen and kidney up to 24 weeks after administration, with full correction of GAG accumulation in those organs. See Jung et al., Mol Cells. 2010; 30(1):13-8. IDS activity in the brain was only increased in the group of animals sacrificed at short-term (6 weeks post-injection); however, this was not sufficient to normalize GAG content in the CNS. See Jung et al., Mol Cells. 2010; 30(1):13-8.
None of aforementioned approaches has fully restored Iduronate-2-sulfatase activity, achieved full eradication of intracytoplasmic inclusions in the CNS and somatic tissues, or corrected all clinical signs of MPSII. Thus, there is a need for novel approaches to the treatment of MPSII that have better efficacy and safety profiles.